Multiplexed KRAS mutation detection assay

ABSTRACT

Provided herein is reagent mixture comprising multiplexed amplification reagents and flap assay reagents for detecting, in a single reaction, mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T, 35A, 35C, 35T or 38A point mutations. Methods that employ the reagent mix and kits for performing the same are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/811,266, filed on Jul. 28, 2015, which is a continuation of U.S.patent application Ser. No. 13/594,674, now issued as U.S. Pat. No.9,127,318, which claims priority to the filing date of U.S. ProvisionalPatent Application Ser. No. 61/548,639 filed Oct. 18, 2011; whichapplications are herein incorporated by reference.

BACKGROUND

Germline KRAS mutations have been found to be associated with Noonansyndrome (Schubbert et al. Nat. Genet. 2006 38: 331-6) andcardio-facio-cutaneous syndrome (Niihori et al. Nat. Genet. 2006 38:294-6). Likewise, somatic KRAS mutations are found at high rates inleukemias, colorectal cancer (Burmer et al. Proc. Natl. Acad. Sci. 198986: 2403-7), pancreatic cancer (Almoguera et al. Cell 1988 53: 549-54)and lung cancer (Tam et al. Clin. Cancer Res. 2006 12: 1647-53). Methodsfor the detection of point mutations in KRAS may be used, for example,to provide a diagnostic for cancer and other diseases.

SUMMARY

Provided herein is reagent mixture comprising multiplexed amplificationreagents and flap assay reagents for detecting, in a single reaction,mutant copies of the KRAS gene that contain any of the 34A, 34C, 34T,35A, 35C, 35T or 38A point mutations. Methods that employ the reagentmix and kits for performing the same are also provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 schematically illustrates some of the general principles of aflap assay.

FIG. 2 schematically illustrates some of the general principles or oneaspect of the subject method.

FIG. 3 schematically illustrates some of the general principles of anexample of a subject assay.

FIG. 4 shows standard curves for both KRAS mutation calibrators, 35Creporting to HEX (Yellow; bottom line)) and 38A reporting to FAM (Green;top line)), and the ACTB calibrator reporting to Quasar 670 (Red; middleline), show good linearity across 5-logs, from 100,000 copies perreaction to 10 copies per reaction. All three markers show similarslopes and intercept values.

FIG. 5 is a graph showing the distribution of percent mutation by sampletype.

FIG. 6 shows tables 3-6.

FIG. 7 shows the oligonucleotides used for multiplex detection andquantification of the seven mutant alleles of KRAS and the ACTB (betaactin) internal control. From top to bottom, SEQ ID NO: 30, SEQ ID NO:17, SEQ ID NO: 22, SEQ ID NO: 4 (left), SEQ ID NO: 8 (right), SEQ ID NO:23, SEQ ID NO: 15, SEQ ID NO: 5 (left), SEQ ID NO: 8 (right), SEQ ID NO:16, SEQ ID NO: 24, SEQ ID NO: 6 (left), SEQ ID NO: 8 (right), SEQ ID NO:13, SEQ ID NO: 25, SEQ ID NO: 1 (left), SEQ ID NO: 8 (right), SEQ ID NO:11, SEQ ID NO: 26, SEQ ID NO: 2 (left), SEQ ID NO: 8 (right), SEQ ID NO:12, SEQ ID NO: 27, SEQ ID NO: 3 (left), SEQ ID NO: 8 (right), SEQ ID NO:14, SEQ ID NO: 28, SEQ ID NO: 7 (left), SEQ ID NO: 8 (right), SEQ ID NO:18, SEQ ID NO: 29, SEQ ID NO: 9 (left), SEQ ID NO: 10 (right), SEQ IDNO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

DEFINITIONS

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in liquid form,containing one or more analytes of interest.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, are functionalized as ethers, amines, or the likes.

The term “nucleic acid” and “polynucleotide” are used interchangeablyherein to describe a polymer of any length, e.g., greater than about 2bases, greater than about 10 bases, greater than about 100 bases,greater than about 500 bases, greater than 1000 bases, up to about10,000 or more bases composed of nucleotides, e.g., deoxyribonucleotidesor ribonucleotides, and may be produced enzymatically or synthetically(e.g., PNA as described in U.S. Pat. No. 5,948,902 and the referencescited therein) which can hybridize with naturally occurring nucleicacids in a sequence specific manner analogous to that of two naturallyoccurring nucleic acids, e.g., can participate in Watson-Crick basepairing interactions. Naturally-occurring nucleotides include guanine,cytosine, adenine and thymine (G, C, A and T, respectively).

The term “nucleic acid sample,” as used herein denotes a samplecontaining nucleic acid.

The term “target polynucleotide,” as used herein, refers to apolynucleotide of interest under study. In certain embodiments, a targetpolynucleotide contains one or more target sites that are of interestunder study.

The term “oligonucleotide” as used herein denotes a single strandedmultimer of nucleotides of from about 2 to 200 nucleotides.Oligonucleotides may be synthetic or may be made enzymatically, and, insome embodiments, are 10 to 50 nucleotides in length. Oligonucleotidesmay contain ribonucleotide monomers (i.e., may be oligoribonucleotides)or deoxyribonucleotide monomers. An oligonucleotide may be 10 to 20, 11to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 80 to 100, 100to 150 or 150 to 200 nucleotides in length, for example.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The term “primer” as used herein refers to an oligonucleotide that has anucleotide sequence that is complementary to a region of a targetpolynucleotide. A primer binds to the complementary region and isextended, using the target nucleic acid as the template, under primerextension conditions. A primer may be in the range of about 15 to about50 nucleotides although primers outside of this length may be used. Aprimer can be extended from its 3′ end by the action of a polymerase. Anoligonucleotide that cannot be extended from it 3′ end by the action ofa polymerase is not a primer.

The term “extending” as used herein refers to any addition of one ormore nucleotides to the end of a nucleic acid, e.g. by ligation of anoligonucleotide or by using a polymerase.

The term “amplifying” as used herein refers to generating one or morecopies of a target nucleic acid, using the target nucleic acid as atemplate.

The term “denaturing,” as used herein, refers to the separation of anucleic acid duplex into two single strands.

The terms “determining”, “measuring”, “evaluating”, “assessing,”“assaying,” “detecting,” and “analyzing” are used interchangeably hereinto refer to any form of measurement, and include determining if anelement is present or not. These terms include both quantitative and/orqualitative determinations. Assessing may be relative or absolute.“Assessing the presence of” includes determining the amount of somethingpresent, as well as determining whether it is present or absent.

The term “using” has its conventional meaning, and, as such, meansemploying, e.g., putting into service, a method or composition to attainan end.

As used herein, the term “T_(m)” refers to the melting temperature of anoligonucleotide duplex at which half of the duplexes remain hybridizedand half of the duplexes dissociate into single strands. The T_(m) of anoligonucleotide duplex may be experimentally determined or predictedusing the following formula T_(m)=81.5+16.6(log₁₀[Na⁺])+0.41 (fractionG+C)−(60/N), where N is the chain length and [Na⁺] is less than 1 M. SeeSambrook and Russell (2001; Molecular Cloning: A Laboratory Manual,3^(rd) ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., ch. 10).Other formulas for predicting T_(m) of oligonucleotide duplexes existand one formula may be more or less appropriate for a given condition orset of conditions.

As used herein, the term “T_(m)-matched” refers to a plurality ofnucleic acid duplexes having T_(m)s that are within a defined range,e.g., within 5° C. or 10° C. of each other.

As used herein, the terms “reaction mixture” and “reagent mixture”refers to an aqueous mixture of reagents that are capable of reactingtogether to produce a product in appropriate external conditions over aperiod of time. A reaction mixture may contain PCR reagents and flapcleavage reagents, for example.

The term “mixture”, as used herein, refers to a combination of elements,that are interspersed and not in any particular order. A mixture isheterogeneous and not spatially separable into its differentconstituents. Examples of mixtures of elements include a number ofdifferent elements that are dissolved in the same aqueous solution, or anumber of different elements attached to a solid support at random or inno particular order in which the different elements are not spatiallydistinct. A mixture is not addressable. To illustrate by example, anarray of spatially separated surface-bound polynucleotides, as iscommonly known in the art, is not a mixture of surface-boundpolynucleotides because the species of surface-bound polynucleotides arespatially distinct and the array is addressable.

As used herein, the term “PCR reagents” refers to all reagents that arerequired for performing a polymerase chain reaction (PCR) on a template.As is known in the art, PCR reagents essentially include a first primer,a second primer, a thermostable polymerase, and nucleotides. Dependingon the polymerase used, ions (e.g., Mg²⁺) may also be present. PCRreagents may optionally contain a template from which a target sequencecan be amplified.

As used herein, the term “flap assay” refers to an assay in which a flapoligonucleotide is cleaved in an overlap-dependent manner by a flapendonuclease to release a flap that is then detected. The principles offlap assays are well known and described in, e.g., Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447). Forthe sake of clarity, certain reagents that are employed in a flap assayare described below. The principles of a flap assay are illustrated inFIG. 1. In the flap assay shown in FIG. 1, an invasive oligonucleotide 2and flap oligonucleotide 4 are hybridized to target 6 to produce a firstcomplex 8 that contains a nucleotide overlap at position 10. Firstcomplex 8 is a substrate for flap endonuclease. Flap endonuclease 12cleaves flap oligonucleotide 4 to release a flap 14 that hybridizes withFRET cassette 16 that contains a quencher “Q” and a nearby quenchedflourophore “R” that is quenched by the quencher Q. Hybridization offlap 14 to FRET cassette 16 results in a second complex 18 that containsa nucleotide overlap at position 20. The second complex is also asubstrate for flap endonuclease. Cleavage of FRET cassette 16 by flapendonuclease 12 results in release of the fluorophore 22, which producesa fluorescent signal. These components are described in greater detailbelow.

As used herein, the term “invasive oligonucleotide” refers to anoligonucleotide that is complementary to a region in a target nucleicacid. The 3′ terminal nucleotide of the invasive oligonucleotide may ormay not base pair a nucleotide in the target (e.g., which may be thesite of a SNP or a mutation, for example).

As used herein, the term “flap oligonucleotide” refers to anoligonucleotide that contains a flap region and a region that iscomplementary to a region in the target nucleic acid. The targetcomplementary regions on the invasive oligonucleotide and the flapoligonucleotide overlap by a single nucleotide such that, when they areannealed to the target nucleic acid, the complementary sequencesoverlap. As is known, if: a) the 3′ terminal nucleotide of the invasivenucleotide and b) the nucleotide that overlaps with that nucleotide inthe flap oligonucleotide both base pair with a nucleotide in the targetnucleic acid, then a particular structure is formed. This structure is asubstrate for an enzyme, defined below as a flap endonuclease, thatcleaves the flap from the target complementary region of the flapoligonucleotide. If the 3′ terminal nucleotide of the invasiveoligonucleotide does not base pair with a nucleotide in the targetnucleic acid, or if the overlap nucleotide in the flap oligononucleotidedoes not base pair with a nucleotide in the target nucleic acid, thecomplex is not a substrate for the enzyme and there is little or nocleavage.

The term “flap endonuclease” or “FEN” for short, as used herein, refersto a class of nucleolytic enzymes that act as structure specificendonucleases on DNA structures with a duplex containing a singlestranded 5′ overhang, or flap, on one of the strands that is displacedby another strand of nucleic acid, i.e., such that there are overlappingnucleotides at the junction between the single and double-stranded DNA.FENs catalyze hydrolytic cleavage of the phosphodiester bond at thejunction of single and double stranded DNA, releasing the overhang, orthe flap. Flap endonucleases are reviewed by Ceska and Savers (TrendsBiochem. Sci. 1998 23:331-336) and Liu et al (Annu. Rev. Biochem. 200473: 589-615). FENs may be individual enzymes, multi-subunit enzymes, ormay exist as an activity of another enzyme or protein complex, e.g., aDNA polymerase. A flap endonuclease may be thermostable.

As used herein, the term “cleaved flap” refers to a single-strandedoligonucleotide that is a cleavage product of a flap assay.

As used herein, the term “FRET cassette” refers to a hairpinoligonucleotide that contains a fluorophore moiety and a nearby quenchermoiety that quenches the fluorophore. Hybridization of a cleaved flapwith a FRET cassette produces a secondary substrate for the flapendonuclease. Once this substrate is formed, the 5′fluorophore-containing base is cleaved from the cassette, therebygenerating a fluorescence signal.

As used herein, the term “flap assay reagents” refers to all reagentsthat are required for performing a flap assay on a substrate. As isknown in the art, flap assays include an invasive oligonucleotide, aflap oligonucleotide, a flap endonuclease and a FRET cassette, asdescribed above. Flap assay reagents may optionally contain a target towhich the invasive oligonucleotide and flap oligonucleotide bind.

As used herein, the term “genomic locus” refers to a defined region in agenome. A genomic locus exists at the same location in the genomes ofdifferent cells from the same individual, or in different individuals. Agenomic locus in one cell or individual may have a nucleotide sequencethat is identical or very similar (i.e., more than 99% identical) to thesame genomic locus in a different cell or individual. The difference innucleotide sequence between the same locus in different cells orindividuals may be due to one or more nucleotide substitutions. A SNP(single nucleotide polymorphism) is one type of point mutation thatoccurs at the same genomic locus between different individuals in apopulation. Point mutations may be somatic in that they occur betweendifferent cells in the same individual. A genomic locus mutation may bedefined by genomic coordinates, by name, or using a symbol.

As used herein, a “site of a mutation” refers to the position of anucleotide substitution in a genomic locus. Unless otherwise indicated,the site of a mutation in a nucleic acid can have a mutant allele orwild type allele of a mutation. The site of a mutation may be defined bygenomic coordinates, or coordinates relative to the start codon of agene (e.g., in the case of the “KRAS G35T mutation”).

As used herein, the term “point mutation” refers to the identity of thenucleotide present at a site of a mutation in the mutant copy of agenomic locus. The nucleotide may be on either strand of a doublestranded DNA molecule.

As used herein, the term “wild type”, with reference to a genomic locus,refers to the alleles of a locus that contain a wild type sequence. Inthe case of a locus containing a SNP, the wild type sequence may containthe predominant allele of the SNP.

As used herein, the term “mutant”, with reference to a genomic locus,refers to the alleles of a locus that contain a mutant sequence. In thecase of a locus containing a SNP, the mutant sequence may contain aminor allele of the SNP. The mutant allele of a genomic locus maycontain a nucleotide substitution that is not silent in that it eitheralters the expression of a protein or changes the amino acid sequence ofa protein, which causes a phenotypic change (e.g., a cancer-relatedphenotype) in the cells that are heterozygous or homozygous for themutant sequence relative to cells containing the wild type sequence.Alternatively, the mutant allele of a genomic locus may contain anucleotide substitution that is silent.

As used herein, the term “corresponds to” and grammatical equivalentsthereof in the context of, for example, a nucleotide in anoligonucleotide that corresponds to a site of a mutation, is intended toidentify the nucleotide that is correspondingly positioned relative to(i.e., positioned across from) a site of a mutation when two nucleicacids (e.g., an oligonucleotide and genomic DNA containing the mutation)are hybridized. Again, unless otherwise indicated (e.g., in the case ofa nucleotide that “does not base pair” or “base pairs” with a pointmutation) a nucleotide that corresponds to a site of a mutation may basepair with either the mutant or wild type allele of a sequence.

As used herein, the term “KRAS” refers to the human cellular homolog ofa transforming gene isolated from the Kirsten rat sarcoma virus, asdefined by NCBI's OMIM database entry 190070.

A sample that comprises “both wild type copies of the KRAS gene andmutant copies of the KRAS gene” and grammatical equivalents thereof,refers to a sample that contains multiple DNA molecules of the samegenomic locus, where the sample contains both wild type copies of thegenomic locus (which copies contain the wild type allele of the locus)and mutant copies of the same locus (which copies contain the mutantallele of the locus). In this context, the term “copies” is not intendedto mean that the sequences were copied from one another. Rather, theterm “copies” in intended to indicate that the sequences are of the samelocus in different cells or individuals.

As used herein the term “nucleotide sequence” refers to a contiguoussequence of nucleotides in a nucleic acid. As would be readily apparent,number of nucleotides in a nucleotide sequence may vary greatly. Inparticular embodiments, a nucleotide sequence (e.g., of anoligonucleotide) may be of a length that is sufficient for hybridizationto a complementary nucleotide sequence in another nucleic acid. In theseembodiments, a nucleotide sequence may be in the range of at least 10 to50 nucleotides, e.g., 12 to 20 nucleotides in length, although lengthsoutside of these ranges may be employed in many circumstances.

As used herein the term “fully complementary to” in the context of afirst nucleic acid that is fully complementary to a second nucleic acidrefers to a case when every nucleotide of a contiguous sequence ofnucleotides in a first nucleic acid base pairs with a complementarynucleotide in a second nucleic acid. As will be described below, anucleic acid may be fully complementary to another sequence “with theexception of a single base mismatch”, meaning that the sequences areotherwise fully complementary with the exception of a single basemismatch (i.e., a single nucleotide that does not base pair with thecorresponding nucleotide in the other nucleic acid).

As used herein the term a “primer pair” is used to refer to two primersthat can be employed in a polymerase chain reaction to amplify a genomiclocus. A primer pair may in certain circumstances be referred to ascontaining “a first primer” and “a second primer” or “a forward primer”and “a reverse primer”. Use of any of these terms is arbitrary and isnot intended to indicate whether a primer hybridizes to a top strand orbottom strand of a double stranded nucleic acid.

The nucleotides of an oligonucleotide may be designated by theirposition relative to the 3′ terminal nucleotide of an oligonucleotide.For example, the nucleotide immediately 5′ to the 3′ terminal nucleotideof an oligonucleotide is at the “−1” position, the nucleotideimmediately 5′ to the nucleotide at the −1 position is the “−2”nucleotide, and so on. Nucleotides that are “within 6 bases” of a 3′terminal nucleotide are at the −1, −2, −3, −4, −5 and −6 positionsrelative to the 3′ terminal nucleotide.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Before the present invention is described in greater detail, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order which is logically possible.

In the following description, the skilled artisan will understand thatany of a number of polymerases and flap endonucleases could be used inthe methods, including without limitation, those isolated fromthermostable or hyperthermostable prokaryotic, eukaryotic, or archaealorganisms. The skilled artisan will also understand that the enzymesthat are used in the method, e.g., polymerase and flap endonuclease,include not only naturally occurring enzymes, but also recombinantenzymes that include enzymatically active fragments, cleavage products,mutants, and variants of wild type enzymes.

In further describing the method, the reagent mixture used in the methodwill be described first, followed by a description of the reactionconditions that may be used in the method.

Reagent Mixtures

A reagent mixture is provided. In certain embodiments, the reagentmixture comprises: a) amplification reagents comprising a thermostablepolymerase, nucleotides, a set of at least seven forward primers, and areverse primer, wherein: i. the 3′ terminal nucleotide of each forwardprimer of the set base pairs with a different point mutation in the KRASgene relative to other forward primers in the set, wherein the pointmutation is selected from the following point mutations: 34A, 34C, 34T,35A, 35C, 35T and 38A; ii. each of the forward primers comprises anucleotide sequence that is fully complementary to a sequence in theKRAS gene with the exception of a single base mismatch within 6 bases ofthe 3′ terminal nucleotide; and iii. each of the forward primers, incombination with the reverse primer, selectively amplifies a differentallele of a KRAS gene, wherein the allele that is amplified is definedby the point mutation to which the 3′ terminal nucleotide base pairs;and b) flap assay reagents comprising a flap endonuclease, a first FRETcassette that produces a fluorescent signal when cleaved, the set of atleast seven forward primers, and a corresponding set of at least sevendifferent flap oligonucleotides that each comprise a nucleotide thatbase pairs with one of the point mutations; wherein the reagent mixtureis characterized in that, when the reagent mixture combined with anucleic acid sample that comprises at least a 1,000-fold excess of wildtype copies of the KRAS gene relative to mutant copies of the KRAS genethat contain one of the point mutations and thermocycled, the reagentmixture can amplify and detect the presence of the mutant copies of theKRAS gene in the sample. The reaction mixture is characterized in thatit can amplify and detect the presence of mutant copies of the KRAS genein the sample. The forward primers of the amplification reagents areemployed as an invasive primer in the flap assay reagents.

The exact identities and concentrations of the reagents present in thereaction mixture may vary greatly but may be similar to or the same asthose independently employed in PCR and flap cleavage assays, with theexception that the reaction mixture may contain Mg²⁺ at a concentrationthat is higher than employed in conventional PCR reaction mixtures(which contain Mg²⁺ at a concentration of between about 1.8 mM and 3mM). In certain embodiments, the reaction mixture described hereincontains Mg²⁺ at a concentration of in the range of 4 mM to 10 mM, e.g.,6 mM to 9 mM. Exemplary reaction buffers and DNA polymerases that may beemployed in the subject reaction mixture include those described invarious publications (e.g., Ausubel, et al., Short Protocols inMolecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrook et al.,Molecular Cloning: A Laboratory Manual, Third Edition, 2001 Cold SpringHarbor, N.Y.). Reaction buffers and DNA polymerases suitable for PCR maybe purchased from a variety of suppliers, e.g., Invitrogen (Carlsbad,Calif.), Qiagen (Valencia, Calif.) and Stratagene (La Jolla, Calif.).Exemplary polymerases include Taq, Pfu, Pwo, UlTma and Vent, althoughmany other polymerases may be employed in certain embodiments. Guidancefor the reaction components suitable for use with a polymerase as wellas suitable conditions for their use is found in the literature suppliedwith the polymerase. Primer design is described in a variety ofpublications, e.g., Diffenbach and Dveksler (PCR Primer, A LaboratoryManual, Cold Spring Harbor Press 1995); R. Rapley, (The Nucleic AcidProtocols Handbook (2000), Humana Press, Totowa, N.J.); Schena and Kwoket al., Nucl. Acid Res. 1990 18:999-1005). Primer and probe designsoftware programs are also commercially available, including withoutlimitation, Primer Detective (ClonTech, Palo Alto, Calif.), Lasergene(DNASTAR, Inc., Madison, Wis.), and Oligo software (NationalBiosciences, Inc., Plymouth, Minn.) and iOligo (Caesar Software,Portsmouth, N.H).

Exemplary flap cleavage assay reagents are found in Lyamichev et al.(Nat. Biotechnol. 1999 17:292-296), Ryan et al (Mol. Diagn. 19994:135-44) and Allawi et al (J Clin Microbiol. 2006 44: 3443-3447).Appropriate conditions for flap endonuclease reactions are either knownor can be readily determined using methods known in the art (see, e.g.,Kaiser et al., J. Biol. Chem. 274:21387-94, 1999). Exemplary flapendonucleases that may be used in the method include, withoutlimitation, Thermus aquaticus DNA polymerase I, Thermus thermophilus DNApolymerase I, mammalian FEN-1, Archaeoglobus fulgidus FEN-1,Methanococcus jannaschii FEN-1, Pyrococcus furiosus FEN-1,Methanobacterium thermoautotrophicum Thermus thermophilus FEN-1,CLEAVASE™ (Third Wave, Inc., Madison, Wis.), S. cerevisiae RTH1, S.cerevisiae RAD27, Schizosaccharomyces pombe rad2, bacteriophage T5 5′-3′exonuclease, Pyroccus horikoshii FEN-1, human exonuclease 1, calf thymus5′-3′ exonuclease, including homologs thereof in eubacteria, eukaryotes,and archaea, such as members of the class II family ofstructure-specific enzymes, as well as enzymatically active mutants orvariants thereof. Descriptions of cleaving enzymes can be found in,among other places, Lyamichev et al., Science 260:778-83, 1993; Eis etal., Nat. Biotechnol. 19:673-76, 2001; Shen et al., Trends in Bio. Sci.23:171-73, 1998; Kaiser et al. J. Biol. Chem. 274:21387-94, 1999; Ma etal., J. Biol. Chem. 275:24693-700, 2000; Allawi et al., J. Mol. Biol.328:537-54, 2003; Sharma et al., J. Biol. Chem. 278:23487-96, 2003; andFeng et al., Nat. Struct. Mol. Biol. 11:450-56, 2004.

As noted above, the reaction mix contains reagents for assaying for, ina single vessel, seven different targets mutations in the KRAS gene. Assuch, the reaction mix contains multiple forward primers (the 3′ basesof each of which base pairs with one of the seven point mutations), asingle reverse primer, multiple different flap oligonucleotides thateach have a nucleotide that base pairs with a single point mutation, andat least one FRET cassette for detecting flap cleavage. In oneembodiment, flap oligonucleotides in a mixture may have a common flap toallow for, for example, the production of the same single fluorescentsignal if any of the seven flap oligonucleotides is cleaved. In anotherembodiment, the flap assay reagents comprise a first FRET cassette and asecond FRET cassette that produce distinguishable fluorescent signalswhen cleaved, and at least one of the at least seven different flapoligonucleotides comprises a flap sequence that hybridizes to the firstFRET cassette and the remainder of said at least seven different flapoligonucleotides hybridizes to the second FRET cassette. In theseembodiments, one fluorescent signal will indicate that one of the subsetof the mutations is present, whereas the other fluorescent signal willindicate that one of the other mutations is present.

In certain cases the reagent mixture may contain a PCR primer pair, aflap oligonucleotide and FRET cassette for the detection of an internalcontrol. In these embodiments, the reaction mixture may further comprisesecond amplification reagents and second flap reagents for amplifyingand detecting a control sequence that is in a gene that is not in KRAS,wherein said second flap reagents comprise a second FRET cassette thatproduces a signal that is distinguishable from the signal of the firstFRET cassette. In particular cases, the control gene may be β-actin,although any suitable sequence may be used.

Upon cleavage of the FRET cassettes, multiple distinguishablefluorescent signals may be observed. The fluorophore may be selectedfrom, e.g., 6-carboxyfluorescein (FAM), which has excitation andemission wavelengths of 485 nm and 520 nm respectively, Redmond Red,which has excitation and emission wavelengths of 578 nm and 650 nmrespectively and Yakima Yellow, which has excitation and emissionwavelengths of 532 nm and 569 nm respectively, and Quasor670 which hasexcitation and emission wavelengths of 644 nm and 670 nm respectively,although many others could be employed.

As noted above, seven of the PCR primers (arbitrarily designated as the“forward” primers), comprises a 3′ terminal nucleotide that base pairswith a point mutation (i.e., a mutant allele) in the genomic locus andalso comprises a nucleotide sequence that is fully complementary to asequence in the locus with the exception of a single base mismatchwithin 6 bases of the 3′ terminal nucleotide (e.g., at the −1 position,the −2 position, the −3 position, the −4 position, the −5 position orthe −6 position, relative to the 3′ terminal nucleotide). In otherwords, in addition to having a 3′ terminal nucleotide that base pairswith only the mutant allele of the mutation in the genomic locus, theprimer also has a destabilizing mismatch near the 3′ end that neitherbases pairs with the mutant allele or the wild type allele of thegenomic region. The mismatch may be at the same or different positionsin each of the forward primers. Without being limited to any particulartheory, the destabilizing mismatch is believed to destabilizehybridization of the 3′ end of the first primer to the wild-typesequence to a greater extend than mutant sequence, thereby resulting inpreferential amplification of the mutant sequence. As will be describedin greater detail below, the presence of the product amplified using thefirst and second primers may be detected using a flap assay that employsthe first primer or another oligonucleotide that has the destabilizingmutation and a terminal nucleotide that base pairs with only the mutantallele at the genomic locus. The use of such a sequence (i.e., asequence that contains the destabilizing mutation and a terminalnucleotide that base pairs with only the mutant allele at the genomiclocus) in the detection step provides further discrimination betweenmutant and wild type sequences in the amplification products. Withoutbeing bound to any particular theory, it is believed that thediscrimination between mutant and wild type largely occurs in the firstfew rounds of amplification since the amplified sequence (i.e., theamplicon) provides a perfectly complementary sequence for the PCRprimers to hybridize to. The wild type sequence should not be amplified,whereas the mutant sequence should be efficiently amplified. The lengthof the nucleotide sequence that is complementary to the KRAS gene in theforward primers may be at least 16 nucleotides in length (e.g., at least17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, to atleast 10 nucleotides or more, in length).

The destabilizing mismatch can be done by substituting a nucleotide thatbase pairs with the point mutation with another nucleotide. Thenucleotide that is substituted into the sequence may be another naturalnucleotide (e.g., dG, dA, dT or dC), or, in certain circumstances, amodified nucleotide. In certain embodiments, the 3′ end of the firstprimer may contain more than 1, e.g., 2 or 3, mismatches. In particularembodiments, the type of mismatch (e.g., whether the mismatch is a G:Tmismatch or a C:T mismatch, etc.) used affects a primer's ability todiscriminate between wild type and mutant sequences. In general terms,the order of the stabilities (from most stable to least stable) ofvarious mismatches are as follows:G:T>G:G=A:G>T:G>G:A=T:T>T:C>A:C>C:T>A:A>C:A>C:C (as described in Gaffneyand Jones (Biochemistry 1989 26: 5881-5889)), although the basepairsthat surround the mismatch can affect this order in certaincircumstances (see, e.g., Ke et al Nucleic Acids Res. 199321:5137-5143). The mismatch used may be optimized experimentally toprovide the desired discrimination.

As would be apparent, the various oligonucleotides used in the methodare designed so as to not interfere with each other. For example, inparticular embodiments, the flap oligonucleotide may be capped at its 3′end, thereby preventing its extension. Further, in certain cases, theT_(m)s of the flap portion of the flap oligonucleotide and the targetcomplementary regions of the flap oligonucleotide may independently beat least 10° C. lower (e.g., 10-20° C. lower) than the T_(m)s of the PCRprimers, which results in a) less hybridization of the flapoligonucleotide to the target nucleic acid at higher temperatures (65°C. to 75° C.) and b) less hybridization of any cleaved flap to the FRETcassette at higher temperatures (65° C. to 75° C.), thereby allowing thegenomic locus to be amplified by PCR at a temperature at which the flapdoes not efficiently hybridize.

In particular cases, the forward primers used for detection of the KRASmutations may have at least 12 contiguous nucleotides (e.g. at least 13,14, 15, 16, 17 or 18 contiguous nucleotides) starting from the 3′ end ofthe following sequences: ACTTGTGGTAGTTGGAGCTCA (SEQ ID NO: 1),ACTTGTGGTAGTTGGAGCTCT (SEQ ID NO: 2), AACTTGTGGTAGTTGGAGATGC (SEQ ID NO:3), CTTGTGGTAGTTGGAGCCA (SEQ ID NO: 4), CTTGTGGTAGTTGGAGCCT (SEQ ID NO:5), TATAAACTTGTGGTAGTTGGACCTC (SEQ ID NO: 6), TGGTAGTTGGAGCTGGTAA (SEQID NO: 7). The flap probe may in certain cases base pair with 10 to 14contiguous nucleotides, e.g., 11 to 13 contiguous nucleotides, of theKRAS gene.

In a multiplex reaction, the primers may be designed to have similarthermodynamic properties, e.g., similar T_(m)s, G/C content, hairpinstability, and in certain embodiments may all be of a similar length,e.g., from 18 to 30 nt, e.g., 20 to 25 nt in length. The other reagentsused in the reaction mixture may also be T_(m) matched.

The assay mixture may be present in a vessel, including withoutlimitation, a tube; a multi-well plate, such as a 96-well, a 384-well, a1536-well plate; and a microfluidic device. In certain embodiments,multiple multiplex reactions are performed in the same reaction vessel.Depending on how the reaction is performed, the reaction mixture may beof a volume of 5 μl to 200 μl, e.g., 10 μl to 100 μl, although volumesoutside of this range are envisioned.

In certain embodiments, a subject reaction mix may further contain anucleic acid sample. In particular embodiments, the sample may containgenomic DNA or an amplified version thereof (e.g., genomic DNA amplifiedusing the methods of Lage et al, Genome Res. 2003 13: 294-307 orpublished patent application US20040241658, for example). In exemplaryembodiments, the genomic sample may contain genomic DNA from a mammaliancell, such as, a human, mouse, rat, or monkey cell. The sample may bemade from cultured cells or cells of a clinical sample, e.g., a tissuebiopsy, scrape or lavage or cells of a forensic sample (i.e., cells of asample collected at a crime scene). In particular embodiments, thegenomic sample may be from a formalin fixed paraffin embedded (FFPE)sample.

In particular embodiments, the nucleic acid sample may be obtained froma biological sample such as cells, tissues, bodily fluids, and stool.Bodily fluids of interest include but are not limited to, blood, serum,plasma, saliva, mucous, phlegm, cerebral spinal fluid, pleural fluid,tears, lactal duct fluid, lymph, sputum, cerebrospinal fluid, synovialfluid, urine, amniotic fluid, and semen. In particular embodiments, asample may be obtained from a subject, e.g., a human, and it may beprocessed prior to use in the subject assay. For example, the nucleicacid may be extracted from the sample prior to use, methods for whichare known.

For example, DNA can be extracted from stool from any number ofdifferent methods, including those described in, e.g, Coll et al (J. ofClinical Microbiology 1989 27: 2245-2248), Sidransky et al (Science 1992256: 102-105), Villa (Gastroenterology 1996 110: 1346-1353) and Nollau(BioTechniques 1996 20: 784-788), and U.S. Pat. Nos. 5,463,782,7,005,266, 6,303,304 and 5,741,650. Commercial DNA extraction kits forthe extraction of DNA from stool include the QIAamp stool mini kit(QIAGEN, Hilden, Germany), Instagene Matrix (Bio-Rad, Hercules, Calif.),and RapidPrep Micro Genomic: DNA isolation kit (Pharmacia Biotech Inc.,Piscataway, N.J.), among others.

Method for Sample Analysis

A method of sample analysis that employs the reagent mix is alsoprovided. In certain embodiments, this method comprises: a) subjecting areaction mixture comprising i. the above-summarized reagent mixture andii. a nucleic acid sample that comprises at least a 100-fold excess ofwild type copies of the KRAS gene relative to mutant KRAS gene thatcontain one of the point mutations, to the following thermocyclingconditions: a first set of 5-15 cycles of: i. a first temperature of atleast 90° C.; ii. a second temperature in the range of 60° C. to 75° C.;iii. a third temperature in the range of 65° C. to 75° C.; followed by:a second set of 20-50 cycles of: i. a fourth temperature of at least 90°C.; ii. a fifth temperature that is at least 10° C. lower than thesecond temperature; iii. a sixth temperature in the range of 65° C. to75° C.; wherein no additional reagents are added to the reaction betweenthe first and second sets of cycles and, in each cycle of the second setof cycles, cleavage of a flap probe is measured; and b) detecting thepresence of a mutant copy of KRAS in the nucleic acid sample.

In these embodiments, the reaction mixture may be subject to cyclingconditions in which an increase in the amount of amplified product(indicated by the amount of fluorescence) can be measured in real-time,where the term “real-time” is intended to refer to a measurement that istaken as the reaction progresses and products accumulate. Themeasurement may be expressed as an absolute number of copies or arelative amount when normalized to a control nucleic acid in the sample.Fluorescence can be monitored in each cycle to provide a real timemeasurement of the amount of product that is accumulating in thereaction mixture.

In some embodiments, the reaction mixture may be subjected to thefollowing thermocycling conditions: a first set of 5 to 15 (e.g., 8 to12) cycles of: i. a first temperature of at least 90° C.; ii. a secondtemperature in the range of 60° C. to 75° C. (e.g., 65° C. to 75° C.);iii. a third temperature in the range of 65° C. to 75° C.; followed by:a second set of 20-50 cycles of: i. a fourth temperature of at least 90°C.; ii. a fifth temperature that is at least 10° C. lower than thesecond temperature (e.g., in the range of 50° C. to 55° C.); and iii. asixth temperature in the range of 65° C. to 75° C. No additionalreagents need to be added to the reaction mixture during thethermocycling, e.g., between the first and second sets of cycles. Inparticular embodiments, the thermostable polymerase is not inactivatedbetween the first and second sets of conditions, thereby allowing thetarget to be amplified during each cycle of the second set of cycles. Inparticular embodiments, the second and third temperatures are the sametemperature such that “two step” thermocycling conditions are performed.Each of the cycles may be independently of a duration in the range of 10seconds to 3 minutes, although durations outside of this range arereadily employed. In each cycle of the second set of cycles (e.g., whilethe reaction is in the fifth temperature), a signal generated bycleavage of the flap probe may be measured to provide a real-timemeasurement of the amount of target nucleic acid in the sample.

The method provided herein is a multiplexed invader assay that employsmismatched primers. The subject method may be readily adapted from themethod shown in FIG. 2 by the addition of at least six other primersthat recognize other point mutations in the KRAS gene, as describedabove. With reference to FIG. 2, the method includes amplifying product30 from sample 32 that comprises both wild type copies of the KRAS gene34 and mutant copies of the KRAS gene 36 that have a point mutation 38(e.g., the 34A, 34C, 34T, 35A, 35C, 35T or 38A mutations) relative tothe wild type gene 34, to produce an amplified sample. The amplifying isdone using a forward primer 40 and a second primer 42, where the firstprimer comprises a 3′ terminal nucleotide 44 that base pairs with thepoint mutation and also comprises a nucleotide sequence that is fullycomplementary to a sequence in the locus with the exception of a singlebase mismatch 46 (i.e., a base that is not complementary to thecorresponding base in the target genomic locus) within 6 bases of 3′terminal nucleotide 44. The presence of product 30 in the amplifiedsample is detected using a flap assay that employs the same forwardprimer as an invasive oligonucleotide 48. As shown in FIG. 2, theforward primer 40 is employed as the invasive oligonucleotide 48 in theflap assay. As described above and in FIG. 1, the flap assay relies onthe cleavage of complex 32 that contains a flap oligonucleotide 50,invasive oligonucleotide 48 and product 30 by a flap endonuclease (notshown) to release flap 52. Released flap 52 then hybridizes to FRETcassette 54 to form a second complex that is cleaved by the flapendonuclease to cleave the fluorophore from the complex and generatefluorescent signal 56 that can be measured to indicate the amount ofproduct in the amplified sample. In this embodiment, the presence of afluorescent signal indicates that there are mutant alleles of the KRASgene in the sample.

The amount of product in the sample may be normalized relative to theamount of a control nucleic acid present in the sample, therebydetermining a relative amount of the mutant copies of KRAS in thesample. In some embodiments, the control nucleic acid may be a differentlocus to the genomic locus and, in certain cases, may be detected usinga flap assay that employs an invasive oligonucleotide having a 3′terminal nucleotide that base pairs with the wild type copies of thegenomic locus at the site of the point mutation, thereby detecting thepresence of wild type copies of the genomic locus in said sample. Thecontrol may be measured in parallel with measuring the product in thesame reaction mixture or a different reaction mix. If the control ismeasured in the same reaction mixture, the flap assay may includefurther reagents, particularly a second invasive oligonucleotide, asecond flap probe having a second flap and a second FRET cassette thatproduces a signal that is distinguishable from the FRET cassette used todetect the mutant sequence. In particular embodiments, the reactionmixture may further comprise PCR reagents and flap reagents foramplifying and detecting a second genomic locus or for detecting asecond point mutation in the same genomic locus.

In certain cases, fluorescence indicating the amount of cleaved flap canbe detected by an automated fluorometer designed to perform real-timePCR having the following features: a light source for exciting thefluorophore of the FRET cassette, a system for heating and coolingreaction mixtures and a fluorometer for measuring fluorescence by theFRET cassette. This combination of features, allows real-timemeasurement of the cleaved flap, thereby allowing the amount of targetnucleic acid in the sample to be quantified. Automated fluorometers forperforming real-time PCR reactions are known in the art and can beadapted for use in this specific assay, for example, the ICYCLER™ fromBio-Rad Laboratories (Hercules, Calif.), the Mx3000P™, the MX3005P™ andthe MX4000™ from Stratagene (La Jolla, Calif.), the ABI PRISM™ 7300,7500, 7700, and 7900 Taq Man (Applied Biosystems, Foster City, Calif.),the SMARTCYCLER™, ROTORGENE2000™ (Corbett Research, Sydney, Australia)the GENE XPERT™ System (Cepheid, Sunnyvale, Calif.) and the LIGHTCYCLER™(Roche Diagnostics Corp., Indianapolis, Ind.). The speed of rampingbetween the different reaction temperatures is not critical and, incertain embodiments, the default ramping speeds that are preset onthermocyclers may be employed.

In certain cases, the method may further involve graphing the amount ofcleavage that occurs in several cycles, thereby providing a real timeestimate of the abundance of the nucleic acid target. The estimate maybe calculated by determining the threshold cycle (i.e., the cycle atwhich this fluorescence increases above a predetermined threshold; the“Ct” value or “Cp” value). This estimate can be compared to a control(which control may be assayed in the same reaction mix as the genomiclocus of interest) to provide a normalized estimate. The thermocyclermay also contain a software application for determining the thresholdcycle for each of the samples. An exemplary method for determining thethreshold cycle is set forth in, e.g., Luu-The et al (Biotechniques 200538: 287-293).

A device for performing sample analysis is also provided. In certainembodiments, the device comprises: a) a thermocycler programmed toperform the above-described method and b) a vessel comprising theabove-described reaction mixture.

Kits

Also provided are kits for practicing the subject method, as describedabove. The components of the kit may be present in separate containers,or multiple components may be present in a single container. Inparticular embodiments, a kit may comprise: a) amplification reagentscomprising a thermostable polymerase, nucleotides, a set of at leastseven forward primers, and a reverse primer, wherein: i. the 3′ terminalnucleotide of each forward primer of the set base pairs with a differentpoint mutation in the KRAS gene relative to other forward primers in theset, wherein the point mutation is selected from the following pointmutations: 34A, 34C, 34T, 35A, 35C, 35T and 38A; ii. each of the forwardprimers comprises a nucleotide sequence that is fully complementary to asequence in the KRAS gene with the exception of a single base mismatchwithin 6 bases of the 3′ terminal nucleotide; and iii. each of theforward primers, in combination with the reverse primer, selectivelyamplifies a different allele of a KRAS gene, wherein the allele that isamplified is defined by the point mutation to which the 3′ terminalnucleotide base pairs; and b) flap assay reagents comprising a flapendonuclease, a FRET cassette, the set of at least seven forwardprimers, and a corresponding set of at least seven different flapoligonucleotides that each comprise a nucleotide that base pairs withone of the point mutations. The particulars of these reagents aredescribed above. The kit further comprises PCR and flap reagents foramplification and detection of a control nucleic acid.

In addition to above-mentioned components, the kit may further includeinstructions for using the components of the kit to practice the subjectmethods. The instructions for practicing the subject methods aregenerally recorded on a suitable recording medium. For example, theinstructions may be printed on a substrate, such as paper or plastic,etc. As such, the instructions may be present in the kits as a packageinsert, in the labeling of the container of the kit or componentsthereof (i.e., associated with the packaging or subpackaging) etc. Inother embodiments, the instructions are present as an electronic storagedata file present on a suitable computer readable storage medium, e.g.CD-ROM, diskette, etc. In yet other embodiments, the actual instructionsare not present in the kit, but means for obtaining the instructionsfrom a remote source, e.g. via the internet, are provided. An example ofthis embodiment is a kit that includes a web address where theinstructions can be viewed and/or from which the instructions can bedownloaded. As with the instructions, this means for obtaining theinstructions is recorded on a suitable substrate. In addition to theinstructions, the kits may also include one or more control samples,e.g., positive or negative controls analytes for use in testing the kit.

Utility

The method described finds use in a variety of applications, where suchapplications generally include sample analysis applications in which thepresence of a mutant KRAS gene in a given sample is detected. Inparticular, the above-described methods may be employed to diagnose, topredict a response to treatment, or to investigate a cancerous conditionor another mammalian disease, including but not limited to, a variety ofcancers such as lung adenocarcinoma, mucinous adenoma, ductal carcinomaof the pancreas and colorectal carcinoma, Noonan syndrome, bladdercancer, gastric cancer, cardio-facio-cutaneous syndrome, leukemias,colon cancer, pancreatic cancer and lung cancer, for example.

In some embodiments, a biological sample may be obtained from a patient,and the sample may be analyzed using the method. In particularembodiments, the method may be employed to identify and/or estimate theamount of mutant copies of a genomic locus that are in a biologicalsample that contains both wild type copies of a genomic locus and mutantcopies of the genomic locus that have a point mutation relative to thewild type copies of the genomic locus. In this example, the sample maycontain at least 100 times (e.g., at least 1,000 times, at least 5,000times, at least 10,000 times, at least 50,000 times or at least 100,000times) more wild type copies of the KRAS gene than mutant copies of theKRAS gene.

Since the point mutation in the KRAS gene have a direct association withcancer, e.g., colorectal cancer, the subject method may be employed todiagnose patients with cancer or a pre-cancerous condition (e.g.,adenoma etc.), alone, or in combination with other clinical techniques(e.g., a physical examination, such as, a colonoscopy) or moleculartechniques (e.g., immunohistochemical analysis). For example, resultsobtained from the subject assay may be combined with other information,e.g., information regarding the methylation status of other loci,information regarding rearrangements or substitutions in the same locusor at a different locus, cytogenetic information, information regardingrearrangements, gene expression information or information about thelength of telomeres, to provide an overall diagnosis of cancer or otherdiseases.

In additional embodiments, if a KRAS mutation is detected in a sample,the identity of the mutation in the sample may be determined. This maybe done by, e.g., sequencing part of the KRAS locus in the sample, or byperforming seven separate assays (i.e., using the same reagents, but notin multiplex form) to determine which of the mutations is present.

In one embodiment, a sample may be collected from a patient at a firstlocation, e.g., in a clinical setting such as in a hospital or at adoctor's office, and the sample may be forwarded to a second location,e.g., a laboratory where it is processed and the above-described methodis performed to generate a report. A “report” as described herein, is anelectronic or tangible document which includes report elements thatprovide test results that may include a Ct value, or Cp value, or thelike that indicates the presence of mutant copies of the genomic locusin the sample. Once generated, the report may be forwarded to anotherlocation (which may the same location as the first location), where itmay be interpreted by a health professional (e.g., a clinician, alaboratory technician, or a physician such as an oncologist, surgeon,pathologist), as part of a clinical diagnosis.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

Example 1 Materials and Methods

Colorectal cancer (CRC) is the second leading cause of cancer deaths inthe United States, yet with effective screening it is potentially themost treatable and preventable cancer (JNCI 2010; 102:89, Ann Intern Med2009; 150:1, Ann Intern Med 2008; 149:441).

The aim of this study was to evaluate the performance of the mutationdetection component of an assay by testing a set of colorectal tissuesthat were characterized using standard dideoxynucleotide sequencing. Theassay is designed to detect the common KRAS mutation sequences at Codons12 and 13, which are found in approximately 35% of all colorectal cancertissues. The current assay combines all seven KRAS mutations in a singlereaction. ACTB (beta-actin) is also included in the reaction to confirmsufficient DNA levels and to ratio KRAS against to establish percentmutation.

A multiplexed KRAS assay was designed utilizing QuARTS (QuantitativeAllele-specific Real-time Target and Signal amplification), a highlysensitive technology that combines allele-specific DNA amplificationwith invasive cleavage chemistry to generate signal during eachamplification cycle similar to real-time PCR. The assay, which detectsseven KRAS mutations and the reference gene β-actin, was used to assess87 colorectal tissue samples (52 CRCs, 16 adenomas≥1 cm, and 19 normalepithelia) as determined by Mayo Clinic Pathology. Samples were obtainedby microdissection of fresh frozen tissue biopsies. DNA was extracted byMayo Clinic using a standardized chloroform/phenol methodology. Thegenotypes of each sample were established using dye terminatordideoxynucleotide sequencing in both the forward and reverseorientations. Copy numbers of KRAS mutations and β-actin were determinedby conventional comparison against standard curves. KRAS data arereported as percent mutation and calculated by dividing mutant copies byβ-actin copies and multiplying by 100.

Tissue Sample Excising, Extraction, and Sequencing

Tissue samples were collected from adenoma and primary tumors and normalcolons at the Mayo Clinic with IRB approval. Patients with confirmedneoplasia had been identified by colonoscopy, endoscopy, radiologic,and/or ultrasound studies. Normal colonic tissue samples were collectedfrom colonoscopy negative patients. For the tumors, pathologist examinedthe tissue sections and circled out histologically distinct lesions todirect careful micro-dissection with about 80% purity. DNA was extractedat Mayo Clinic using either the QIAamp DNA Micro Kit (PN 56304Germantown, Md.) or a standardized chloroform/phenol methodology. TissueDNAs were stored at −80° C.

Sequencing

The KRAS genotypes of each cancer or adenoma sample were establishedusing dye terminator dideoxynucleotide sequencing in both the forwardand reverse orientations for a region including codons 12 and 13 of theKRAS gene. Samples were sequenced on an ABI 3730XL DNA Analyzer usingBig Dye Terminator v3.1 reagents (Applied Biosystems). Mutation Surveyorv3.30 software (SoftGenetics) was used to make the calls. Lane qualityscores for the traces were greater than 20, indicating less than 5%average background noise, and base calls were made based on signal tonoise ratios, peak heights, overlap, and drop-off rate. When qualityscores were above 20 in both directions, concurrence in both directionswas required to verify an alteration from wild type (WT). If onedirection was of low quality, but the other was above the threshold of20, the single high quality read was sufficient to make a call. Traceswere manually inspected for accuracy and 2 positive calls were madewhich were below the sensitivity of the software. Normal colon sampleswere not sequenced. Only mutation 34C was not represented in thesesamples.

QuARTS Assay Techniques

DNA samples extracted from tissues were assessed for the presence ofmutations in exon 2 of the KRAS gene (See Table 1) and the referencegene Beta-Actin using a multiplexed QuARTS (Quantitative Allele-specificReal-time Target and Signal amplification) assay.

TABLE 1 KRAS Amino mutation Amino Amino acid in KRAS short Codon acid inacid KRAS Mutation form Location mutation change WT 35G > A 35A Codon 12Aspartate Gly12Asp Glycine 35G > T 35T Codon 12 Valine Gly12Val 34G > T34T Codon 12 Cystine Gly12Cys 35G > C 35C Codon 12 Alanine Gly12Ala34G > A 34A Codon 12 Serine Gly12Ser 34G > C 34C Codon 12 ArginineGly12Arg 38G > A 38A Codon 13 Aspartate Gly13Asp

The assay is specific to the mutant KRAS DNA and is able to discriminatemutants from wild-type with low cross-reactivity. Specificity isachieved by the use of allele specific PCR with specific mismatches inthe forward primer to preferentially amplify mutant alleles combinedwith semi-quantitative invasive cleavage reactions that furtherdiscriminate and detect the amplified target using real-timefluorescence detection.

The QuARTS reaction was optimized so the primers and probes for eachmutation would function properly at same cycling and reaction conditionsallowing all eight markers to be combined in a single reaction. Cyclingconditions are designed to preferentially amplify mutant sequences byusing a higher annealing temperature in the first 10 cycles, followed by35 cycles at lower annealing temperature required for the invasivecleavage reaction. Fluorescent acquisition begins after the first 10cycles. Multiplex KRAS assays were first optimized in a two-dyeconfiguration where all mutations reported to one dye while ACTBreported to a second dye. The assay was further optimized to improvespecificity and sensitivity by moving to a 3-dye configuration. The3-dye KRAS QuARTS multiplex is configured to report to different dyes sothat 4 mutations report to one dye (G35A, G35C, G34A, and G34C), 3report to a second dye (G35T, G34T and G38A), and ACTB reports to athird dye (see FIG. 3).

The KRAS QuARTS multiplex technology generates highly sensitive andspecific signal from mutant KRAS sequences and a beta-actin referencegene by utilizing two simultaneous reactions (FIG. 3). In the firstreaction, allele-specific amplification is achieved with a uniqueforward primer for each mutation in combination with a single KRASreverse primer. Each forward primer contains a double mismatch to theKRAS WT sequence near the 3′ end of the primer, which prevents efficientamplification of KRAS WT, but has only a single mismatch to the mutationsequence. Taq (recombinant Hot Start Go Taq, Promega, Madison, Wis.) isable to extend efficiently through a single mismatch but not through adouble mismatch near the 3′ end. Signal generation occurs in the secondreaction. A target specific probe binds to the mutant amplicon to forman overlap flap substrate. The 5′ flap is then cleaved by the Cleavaseenzyme (Hologic, Madison, Wis.). The flap sequence is complementary to aFRET cassette. Once the flap is cleaved, it binds to the target FRETcassette and causes the release of the fluorophore to generate signal.The seven KRAS probes share two different flap sequences, which reportto either a HEX or FAM FRET cassette. A probe specific to beta-actincontains a third flap sequence and reports to Quasar 670 FRET cassette.The use of 3 different flap sequences that correspond to a FAM, HEX orQuasar 670 FRET cassette allows the assay to distinguish KRAS mutationsfrom the beta-actin reference gene in a single well. In total, the KRASmultiplex QuARTS assay combines seven KRAS forward primers, a singleKRAS reverse primer, 7 KRAS probes, a beta-actin forward and reverseprimer, and three FRET cassettes. The concentration ranges for primersare from 105 to 245 nM, probes are from 90 to 250 nM, and FRET cassettesare at 100 nM each.

Reactions were setup by adding 10 μL of DNA from each sample toappropriate wells of a 96-well plate containing 20 μL of assay-specificQuARTS™ master mix. Each plate was run on an ABI 7500 Fast Dx Real-TimePCR Instrument. Calibrators and controls were included in each run.After the run was completed, data was exported to an Exact Sciencesanalysis template, and the cycle threshold value was calculated as thecycle at which the fluorescent signal per channel for a reaction crossesa threshold of 18% of the maximum fluorescence for that channel DNAstrand number was determined by comparing the cycle threshold of thetarget gene to the calibrator curve for that assay. Calibrators weremade from plasmids with single target inserts, mutation 38A was used forthe FAM channel and 35C was used for the HEX channel Percent mutationwas determined for each marker by dividing KRAS strands by ACTB strandsand multiplying by 100. All mutations reporting to the FAM dye arequantified using KRAS 38A calibrators, and all mutations reporting tothe HEX dye are quantified using KRAS 35C calibrators. The calibratorsfor all three dyes show similar linearity (FIG. 4) and goodreproducibility. The assay was optimized to minimize cross-reactivitywith KRAS WT plasmid at 200,000 strands per reaction, which wasapproximately 0.07 to 0.11 percent mutation in the 3-dye configuration.

Results

The 2-dye KRAS multiplex QuARTS assay was evaluated using 87 tissuesamples consisting of 19 normal, 16 adenoma and 52 colorectal cancersamples. KRAS QuARTS results showed good agreement with sequencing data.All normal colon samples had a value equal to or less than 0.55 percentmutation. All of the samples that were KRAS positive by sequencing weregreater than 8.35 percent mutation. The colorectal cancer and adenomasamples that were KRAS negative by sequencing showed a range from 0.04to 1.33 percent mutation with a mean of 0.4±0.29%. (Tables 3 and 4;shown in FIG. 6).

The 3-dye KRAS multiplex QuARTS assay was evaluated using 191 tissuesamples consisting of 47 normal, 48 adenoma, and 96 colorectal cancersamples (Tables 2 below and Tables 5 and 6 shown in FIG. 6). This setincluded 86 of the 87 tissues that were also tested with the 2-dyeconfiguration (Table 4; shown in FIG. 6). KRAS QuARTS showed excellentagreement with sequencing data. All of the samples that were KRASpositive by sequencing showed at least 2.45 percent mutation in the KRASQuARTS assay, with a mean of 43.73±33.3 percent mutation. The colorectalcancer and adenoma samples that were negative by sequencing showed arange from 0.00 to 1.99 percent mutation with a mean of 0.14±0.33percent mutation. The adenoma sample that showed 1.99 percent mutationwas detected at 1.33 percent mutation in the 2-dye configuration. Allnormal colon samples had a value equal to or less than 0.21 percentmutation (mean for normal samples was 0.03±0.04 percent mutation).

TABLE 2 Sequence and QuARTS assay data concordance: Subset of Tissue DNAAverage ACTB % strands % mutation mutation Sample Colon tissue per FAMHEX name histology reaction Channel channel Genotype TS1 Cancer 110902.45% 1.04% 35A TS2 Adenoma 5187 11.74% 6.30% 35A TS3 Cancer 2180 19.29%0.03% 35T TS4 Adenoma 1172 37.80% 0.02% 35T TS5 Cancer 46798 55.69%0.00% 38A TS6 Cancer 64399 3.46% 61.78% 34A TS7 Adenoma 3349 16.16%19.20% 35A TS8 Cancer 7578 12.60% 0.00% 34T TS9 Adenoma 18667 1.99%0.07% WT TS10 Cancer 52344 0.06% 0.00% WT TS11 Cancer 20977 0.02% 0.00%WT TS12 Adenoma 51648 0.01% 0.52% WT TS13 Adenoma 43957 0.03% 0.01% WTTS14 Adenoma 98260 0.12% 0.30% WT TS15 Normal colon 21484 0.00% 0.00% WTTS16 Normal colon 92492 0.03% 0.00% WT TS17 Normal colon 13228 0.01%0.00% WT TS18 Normal colon 579 0.00% 0.00% WT TS19 Normal colon 2116990.00% 0.18% WT TS20 Normal colon 85889 0.01% 0.02% WT TS21 Adenoma 409763.83% 41.74% 34T; 35C TS22 Cancer 1806 101.96% 0.00% 38A TS23 Adenoma1864 1.37% 43.58% 34A TS24 Adenoma 6433 48.83% 4.70% 35T

The KRAS QuARTS multiplex assay showed a maximum of 0.11 percentmutation for cross-reactivity with KRAS WT plasmid control at 200,000strands per reaction.

FIG. 5 shows the distribution of percent mutation by sample type. Withthe highest normal giving 0.21 percent and the lowest sequencingconfirmed KRAS mutation at 2.45 percent the assay agrees 100% on thosesamples. Because of the higher sensitivity of the QuARTS assay 2 cancersand 12 adenomas are observed that are elevated above the highest percentmutation of the normal samples.

Based on sequencing data: the 52 CRC samples contained 22 KRAS mutationsand 30 wild-type genotypes, the 16 adenomas≥1 cm contained 8 mutationsand 8 wild-type genotypes, and the 19 normal tissues contained allwild-type genotypes. The QuARTS assay detected 100% of the KRASmutations in the CRC and adenomas and provided excellent differentiationbetween wild-type and mutation, with the highest percent KRAS mutationof normal wild-type samples at 0.55% and the lowest percent mutation ofKRAS positive samples at 8.34%. Based on this data, this assay is moresensitive analytically than standard sequencing.

In this study we were able to show results for 6 of the 7 mutationsdetected by the assay; mutation 34C (Gly12Arg) in exon 2 represents 0.5%of KRAS mutations in colorectal cancers and was not represented in thesesamples. Using plasmid derived sequences we have shown the assay iscapable of detecting this mutation (data not shown).

This multiplex does not distinguish among mutations. The assay showssome cross-reactivity between mutations which is likely to improvesensitivity since the signal is increased without any increase in WTcross reactivity.

The three-dye configuration of the KRAS QuARTS multiplex assay showedbetter specificity than the 2-dye version; when all KRAS mutations arereporting to a single dye, the signal from cross-reactivity with WT isadditive but by distributing the KRAS mutation signal across two dyes,the cross-reactivity with WT is reduced by more than half.

Example 2 Materials and Methods

FIG. 7 shows the designs used for multiplex detection and quantificationof the seven mutant alleles of KRAS and the ACTB (beta actin) internalcontrol. Three 5′-flaps (A5 and A7 for KRAS and A1 for ACTB) were usedin the assay. The probes with flaps A5 and A7, used for KRAS mutants,were used in conjunction with two FRET oligonucleotides A5-HEX andA7-FAM thus giving signal in these two dye channels for KRAS mutations.The ACTB probe, on the other hand, had a 5′-flap A1-Quasar, resulting inQuasar 670 signal when ACTB is present. Further details of the reagentmix are set forth below.

Reagent Mix Components

Mutation QuARTS Assay Primers

Conc in final Primer reaction Name Sequence (nM) KRAS RP10GATTCTGAATTAGCTGTATC 350 GT (SEQ ID NO: 8) KRAS 35A ACTTGTGGTAGTTGGAGCTC250 P2C A (SEQ ID NO: 1) KRAS 35T ACTTGTGGTAGTTGGAGCTC 250 P2CT (SEQ ID NO: 2) KRAS 35C AACTTGTGGTAGTTGGAGAT 250 P4A GC (SEQ ID NO: 3)KRAS 34A CTTGTGGTAGTTGGAGCCA 250 P2C 19b (SEQ ID NO: 4) KRAS 34TCTTGTGGTAGTTGGAGCCT 250 P2C (SEQ ID NO: 5) KRAS 34C TATAAACTTGTGGTAGTTGG250 P4C-b ACCTC (SEQ ID NO: 6) KRAS 38A TGGTAGTTGGAGCTGGTAA 250 P2A 19b(SEQ ID NO: 7) ACTB WT CCATGAGGCTGGTGTAAAG 150 FP3 (SEQ ID NO: 9)ACTB WT CTACTGTGCACCTACTTAAT 150 RP3 ACAC (SEQ ID NO: 10)Mutation QuARTS Assay Probes

Conc in final reaction Probes Probe sequence (nM) KRAS 35T A7 PbGCGCGTCCTTGGCGTAGGCA/3C6/ 310 (SEQ ID NO: 11) KRAS 35C A5 PbCCACGGACGCTGGCGTAGGCA/3C6/ 310 (SEQ ID NO: 12) KRAS 35A A5 PbCCACGGACGATGGCGTAGGCA/3C6/ 310 (SEQ ID NO: 13) KRAS 38A A7 PbGCGCGTCCACGTAGGCAAGA/3C6/ 310 (SEQ ID NO: 14) KRAS 34T A7 PbGCGCGTCCTGTGGCGTAGGC/3C6/ 310 (SEQ ID NO: 15) KRAS 34C AS PbCCACGGACGCGTGGCGTAGGC/3C6/ 310 (SEQ ID NO: 16) KRAS 34A AS pbCCACGGACGAGTGGCGTAGGC/3C6/ 310 (SEQ ID NO: 17) ACTB WT Pb4 A1CGCCGAGGGCGGCCTTGGAG/3C6/ 310 (SEQ ID NO: 18)FRET Cassettes—

all FRET sequences exactly match the arm landing pad (no extra 3′ bases)

Conc in final reaction name sequence 5′-3′ (nM) Arm 5 TAMRA FRETTAMRA/TCT/BHQ2/AGCCGGTTTT 100 CCGGCTGAGACGTCCGTGG/3C6/ (SEQ ID NO: 19)Arm 7 FAM FRET FAM/TCT/BHQ1/AGCCGGTTTTCC 100 GGCTGAGAGGACGCGC/3C6/(SEQ ID NO: 20) Arm 1 Quasar 670 Quasar 670/TCT/BHQ2/AGCCG 100 FRETGTTTTCCGGCTGAGACCTCGGCG/ 3C6/ (SEQ ID NO: 21)Other Components

Current reaction buffer components Reagents Concentration per reactionrecombinant HotStart Go Taq 0.07 U/uL water, PM1009 NA PM1143, ElutionBuffer Teknova Te pH 8.0 buffer + 20 ng/uL tRNA dNTPs* 250 μM MOPS 10 mM1M KCl 0.797 mM 2M MgCl2 7.5 mM 5M Tris-HCl pH 8 0.319 mM 50% Tween-200.008% 20% IGEPAL 0.008% 80% glycerol  1.25% Cleavase 7.3 ng/uL BSA 100ng/uL (3 ug/30 uL reaction) *likely to be part of oligo mix rather than20X reaction bufferThermocycling Parameters

Number of Stage Temp/Time Ramp Rate Cycles Pre-incubation 95° C./3′ 100%1 Amplification 1 95° C./20″ 100% 10 64° C./30″ 100% 70° C./30″ 100%Amplification 2 95° C./20″ 100% 35 53° C./1′ 100% 70° C./30″ 100%Cooling 40° C./30″ 100% 1

Results

Using an oligonucleotide mixture with three FRET oligonucleotides (FAMor HEX with Quasar) and one KRAS mutant and ACTB specificoligonucleotide mixture, it was found that signal is generated for ACTBonly when ACTB is present. However, for KRAS, it was found thatcross-reactive signal is generated in HEX and FAM channels between somemutant KRAS mutations (see table below) with minimal cross-reactivitysignal for wild-type KRAS. For example, when the target is KRAS 34Cmutation, the 35A and 35T oligonucleotide mixtures gave appreciablecross-reactive signal. The table below shows the results reported ascycle threshold values obtained using 1,000 copies of the different KRASmutants, and 10,000 copies for ACTB targets with either duplex ormultiplex oligonucleotide mixes. Based on these results, and based onthe low cross reactivity of 35C and 38A targets, those two targets wereselected to be the calibrators used for standard curve generation.

Target: Reporting Oligo 34A 34C 34T 35 35C 35T 38A ACTB WT NTC Dyes mix:1 2 3 4 5 6 7 8 9 10 HEX/Quasar 34A A 13.3 29.6 26.6 33.5 31.7 10.6 25.1HEX/Quasar 34C B 23.1 13.2 19.7 28.4 31.8 10.5 26.4 FAM/Quasar 34T C22.5 20.2 10.8 28.5 34.3 25.7 9.2 21.1 HEX/Quasar 35A D 25.3 13.8 24.512.9 28.4 27.9 31.1 10.4 26.8 HEX/Quasar 35C E 29.4 30.5 19.6 11.6 18.229.9 10.4 26.2 FAM/Quasar 35T F 27.2 14.1 25.3 22.0 13.2 27.6 8.6 26.5FAM/Quasar 38A G 31.7 32.6 32.6 32.0 23.5 10.4 9.4 22.0 26.0 FAM/HEX/All H 18.3/ 13.4/ 12.3/ 13.5/ 22.2/ 12.7/ 13.6/ 29.6/ 25.4/ QuasarMultiplex 11 10 18.6 8.6 11.8 15.3 33 10.5 22.1

When a plasmid containing a triple insert of 2 KRAS mutations (35C &38A) and one ACTB (i.e., 35C/38A/ACTB plasmid), was used as calibrators(i.e. standard curves) to calculate strand numbers for all othermutations, data showing the following was obtained:

-   -   a) Less than 0.05% cross reactivity between the multiplex KRAS        mutant oligonucleotide mixes and wild-type KRAS.    -   b) 35C standard curve calibrator can be used to quantify the        HEX-reporting mutants (35A, 35C, 34A, 34C) and, similarly, that        the 38A standard curve calibrator can be used to quantify the        FAM-reporting mutants (38A, 34T, 35T).    -   c) The assay can be used for both detection (i.e. screening for        mutations) and quantification of KRAS mutants with minimal        cross-reactivity with wild-type.    -   d) The sensitivity of the assay is approximately a single-copy        per reaction.

Additionally, using the multiplex KRAS mutant assay designs forscreening previously sequenced tissue samples by assigning the HEXsignal (35C calibrator) as an indicator for the presence of the 35A,35C, 34A, 34C mutations and the FAM signal (38A calibrator) as anindicator for the presence of the 38A, 34T, 35T mutations the followingresults were obtained:

Test of tissue and stool samples with 3 color KRAS QuARTS assay GenotypeKRAS KRAS ACTB ACTB % % Sample by Colon tissue 35C 38A Strands Strandsmutation mutation ID sequencing histology Strands* Strands* (35C)**(38A)** HEX FAM Call Tissue Samples 054DAA G34T ADENOMA — 6,785 17,99822,346  0% 30% Positive 036DAA G35A CANCER 641 1,061 4,890 5,953 13% 18%Positive 029DAA G34A CANCER 34,833 2,509 56,382 72,416 62%  3% Positive089DAA G35A ADENOMA 126 180 1,821 2,098  7%  9% Positive 056DAA G35TADENOMA 0 485 1,063 1,282  0% 38% Positive 026DAA G38A CANCER — 29,20041,166 52,430  0% 56% Positive 019DAA WT normal colon — — 42,028 51,976 0%  0% Negative 008DAA WT normal colon — 3 39,535 48,897  0%  0%Negative 092DAA WT normal colon — 16 81,065 100,809  0%  0% Negative017DAA WT normal colon — 32 113,608 149,212  0%  0% Negative 013DAA WTnormal colon — 50 99,401 130,064  0%  0% Negative S1  G35T ADENOMA 335,353 18,647 22,351  0% 24% Positive S17 G38A CANCER — 5,227 9,97111,816  0% 44% Positive S40 G34T CANCER — 14,732 13,856 16,462  0% 89%Positive S84 WT normal colon — 6 29,760 36,621  0%  0% Negative S85 WTnormal colon — 1 11,982 14,111  0%  0% Negative S86 WT normal colon — 63161,567 206,770  0%  0% Negative S87 WT normal colon — — 22,006 26,459 0%  0% Negative Stool Samples Genotype KRAS KRAS ACTB ACTB % % Sampleby Colon tissue 35C 38A Strands Strands mutation mutation ID sequencinghistology Strands Strands (35C) (38A) HEX FAM Call S12 38G > A CANCER 415,845 48,028 36,827 0% 33% Positive S11 34G > T CANCER — 2,473 10,6728,410 0% 23% Positive S8  35G > A CANCER 1,156 1,379 173,401 129,130 1% 1% Positive S9  35G > T ADENOMA 122 17,057 59,046 45,326 0% 29%Positive S2  35G > T CANCER 44 8,079 72,221 55,335 0% 11% Positive S7 38G > A CANCER 1 11,441 117,166 88,606 0% 10% Positive S3  35G > TCANCER 2 956 12,573 9,921 0%  8% Positive S21 35G > T ADENOMA 20 47427,373 21,292 0%  2% Positive *Two plasmids, 35C/ACTB and 38A/ACTB, wereused for generation of standard curves for HEX/Quasar and FAM/Quasar,respectively. **ACTB Strands (35C) are calculated based on theACTB/Quasar standard curve generated using the 35C/ACTB plasmid. **ACTBStrands (38A) are calculated based on the ACTB/Quasar standard curvegenerated using the 38A/ACTB plasmid.

The data shows full agreement between QuARTS and sequencing. Thisindicates that the KRAS mutant QuARTS assay can be used on both stoolDNA as well as tissue samples to screen for KRAS mutations.

What is claimed is:
 1. A kit comprising: at least seven differentprimers that each comprise, at their 3′ end, at least 12 contiguousnucleotides starting from the 3′ end of a sequence selected from SEQ IDNOs: 1-7, wherein the primers are in one or more containers.
 2. The kitof claim 1, wherein each primer is in a separate container.
 3. The kitof claim 1, wherein the primers are each complementary to at least 16nucleotides in the human KRAS gene.
 4. The kit of claim 1, wherein theprimers each comprise a sequence selected from SEQ ID NOS: 1-7.
 5. Thekit of claim 1, further comprising a reverse primer that hybridizes tothe human KRAS gene.
 6. The kit of claim 1, wherein the kit furthercomprises a thermostable polymerase.
 7. The kit of claim 1, wherein thekit further comprises nucleotides.
 8. The kit of claim 1, furthercomprising flap assay reagents that comprise a flap endonuclease, a FRETcassette that produces a fluorescent signal when cleaved, and at leastseven different flap oligonucleotides.
 9. The kit of claim 8, whereinthe flap oligonucleotides each base pair with 10 to 14 contiguousnucleotides of the human KRAS gene.
 10. The kit of claim 9, wherein theflap oligonucleotides each comprise at their 3′ ends at least 11contiguous nucleotides starting from the 3′ end of a sequence as setforth in SEQ ID NOs: 11-17.
 11. The kit of claim 1, wherein the kitcomprises: a) amplification reagents comprising a thermostablepolymerase, nucleotides, said at least seven different primers, and areverse primer, wherein each of the seven different primers, incombination with said reverse primer, selectively amplifies a differentallele of a KRAS gene, wherein the allele that is amplified is definedby a point mutation selected from 34A, 34C, 34T, 35A, 35C, 35T and 38A;and b) flap assay reagents comprising a flap endonuclease, a first FRETcassette that produces a fluorescent signal when cleaved, said at leastseven different primers, and a corresponding set of at least sevendifferent flap oligonucleotides that each comprise a nucleotide thatbase pairs with one of the point mutations.
 12. The kit of claim 11,wherein the seven different flap oligonucleotides comprise at their 3′ends at least 11 contiguous nucleotides starting from the 3′ end of asequence as set forth in SEQ ID NOs: 11-17.